Methylmalonic acid, in urine

Biochemically, malonyl-CoA decarboxylase deficiency is characterised by the excretion of excess malonic and methylmalonic acids in urine, mostly... 

Methylmalonyl-CoA mutase is a cobalamin-linked enzyme of mitochondria that catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA. A reduction of this enzyme due to vitamin B12 deficiency will result in a metabolic block with the urinary excretion of methylmalonic acid, and the measurement of this metabolite has been used to confirm a deficiency of vitamin B12. The test has also been useful in investigating rare abnormalities of this enzyme that result in the excretion of methylmalonic acid in the presence of adequate vitamin B12. Given an oral loading dose of valine or isoleucine will increase the urinary excretion of methylmalonic acid in patients with a vitamin B12 deficiency (G4). However, Chanarin and his colleagues (CIO) found that one-quarter of their patients with pernicious anemia excreted a normal concentration of methylmalonic acid even after a loading dose of valine. Normal subjects excrete up to 15 mg of methylmalonic acid in their urine over a 24-hour period (Cll). 

Whlean et al. (W7) described a follow-up, extending over several years, of two infants with methylmalonic aciduria unresponsive to treatment with vitamin B12. The first patient, a boy, was the child of two first cousins delivery followed an uneventful pregnancy. The child had convulsions 4 days after birth and was found to have a profound metabolic acidosis, and was excreting a large amount of methylmalonic acid in his urine. His serum vitamin B12 concentration was normal. Further studies confirmed a diagnosis of methylmalonic aciduria. 

Marsh DB, Nuttall KL. Methylmalonic acid in clinical urine specimens by capillary zone electrophoresis using indirect photometric detection. J Cap Elec 1995 2 63-7. 

S. a,a-Dimethylsuccinic Acid. First found, like methylmalonic acid, in the urine of rats fed a necrogenic diet (F6), a,a-dimethyl-succinic acid was identified later in the urine of normal rats (B3). Its origin is unknown,... 

They are defects in the degradation pathways of leucine, isoleucine, and valine. These conditions are usually diagnosed by examining organic acids in urine with abnormal metabolites also notable on acylcamitine profile. Organic acidemias comprise a variety of disorders and include methylmalonic acidemia (MMA), propionic acidemia (PROP), isovaleric acidemia (IVA), glutaric acidemia type 1 (GA-1), 3-methylcrotonyl carboxylase deficiency (3-MCC), 3-methylglutaconic acidemia (3-MGA), and vitamin B12 uptake, transport, and synthesis defects. 

Vitamin B,2 serves as the coenzyme for some enzymes. One of these is the enzyme that catalyzes the reaction of isomerization of methylmalonic acid, H00CCH(CH3)C00H, to succinic acid. Some children suffer from the disease methylmalonicaciduria, in which the concentration of methylmalonic acid in the blood and urine is very large because of a genetic defect that causes an abnormality in this enzyme and the consequent failure to convert the methylmalonic acid to succinic acid. For about half of the patients the disease can be kept under control by the daily intake of about 1 mg of B12, 500 times the usually recommended intake. The probable explanation of this effect is that the patient manufactures a defective apoenzyme with a very small combination constant with the coenzyme, so that at equilibrium under ordinary nutrition only a very small fraction of the apoenzyme is converted to the active enzyme. It can be seen from Equation 14-4, however, that the amount converted to active enzyme is increased by an increase in the concentration of the coenzyme. 

Although cobalt ions are found in both the (II) and (III) oxidation states, the most important biological compound of cobalt is vitamin B12 or cobalamin where the Co(III) form is present (256) (Fig. 6.10). Cobalamin or related substances are important biological compounds that are involved in a great variety of activities, particularly in bacteria. Vitamin B12 is also necessary in the nutrition of humans and probably of most animal and plant species. It is of critical importance in the reactions by which residues from carbohydrates, fats and proteins, are used to produce energy in living cells. Pernicious anemia is a severe disease in elderly people. This disease is usually accompanied in mammals by the increased excretion of methylmalonic acid in the urine. Today it is effectively controlled by a 100 /ig injection of vitamin B,2. 

In vitamin Bj2 deficiency, methylmalonic acid is excreted in the urine [103]. However, there also exists methylmalonic acidaemia as an inborn error of metabolism, distinct from vitamin B12 deficiency, with higher concentrations of methylmalonic acid in blood and urine [99, 104]. The blood and urine contain excess glycine—up to 12 mg per 100 ml blood and up to 4,000 mg per g creatinine in urine [99, 104-107]. The site of the metabolic block is reaction 4, methylmalonic acidaemia is caused by lack or inactivity of methylmalonyl-CoA mutase [108, 109]. Although there is no deficiency of vitamin Bi2, giving large doses causes a decrease in methylmalonic acid excretion in some cases (Bi2 i esponsive) [110]. Liver biopsy specimens from Bi2 i esponsive patients converted methyl-malonyl-CoA to succinyl-CoA at 10% of the normal rate, rising to normal with added Bj2 cofactor specimens from Bi2-unresponsive patients did not convert any methyl-malonyl-CoA to succinyl-CoA with or without added Bj2 cofactor [111]. 

Magera MJ, Helgeson JK, Matern D, Rinaldo P (2000) Methylmalonic acid measured in plasma and urine by stable-isotope dilution and electrospray tandem mass spectrometry. Clin Chem 46 1804-1810... 

When Rinaldo analyzed Ryan s blood serum, he found high concentrations of methylmalonic acid, a breakdown product of the branched-chain amino acids isoleucine and valine, which accumulates in MMA patients because the enzyme that should convert it to the next product in the metabolic pathway is defective. And particularly telling, he says, the child s blood and urine contained massive amounts of ketones, another metabolic consequence of the disease. Like Shoemaker, he did not find any ethylene glycol in a sample of the baby s bodily fluids. The bottle couldn t be tested, since it had mysteriously disappeared. Ri-naldo s analyses convinced him that Ryan had died from MMA, but how to account for the results from two labs, indicating that the boy had ethylene glycol in his blood Could they both be wrong ... 

Correct answer = B. Alkaptonuria is a rare metabolic disease involving a deficiency in homogentisic acid oxidase, and the subsequent accumulation of homogentisic acid in the urine, which turns dark upon standing. The elevation of methylmalonate (due to methylmalonyl CoA mutase deficiency), phenylpyruvate (due to phenylalanine hydroxlyase deficiency), a-ketoisovalerate (due to branched-chain a-ketoacid dehydrogenase deficiency), and homocystine (due to cystathionine synthase deficiency) are inconsistent with a healthy child with darkening of the urine. 

In this hereditary disease up to 1 - 2 g of methylmalonic acid per day (compared to a normal of <5 mg/day) is excreted in the urine, and a high level of the compound is present in blood. Two causes of the rare disease are known/ One is the lack of functional vitamin B12-containing coenzyme. This can be a result of a mutation in any one of several different genes involved in the synthesis and transport of the cobalamin coenzyme.6 Cultured fibroblasts from patients with this form of the disease contain a very low level of the vitamin B12 coenzyme (Chapter 16), and addition of excess vitamin B12 to the diet may restore coenzyme synthesis to normal. Among elderly patients a smaller increase in methylmalonic acid excretion is a good indicator of vitamin B12 deficiency. A second form of the disease, which does not respond to vitamin B12, arises from a defect in the methylmalonyl mutase protein. Methylmalonic aciduria is often a very severe disease, frequently resulting in death in infancy. Surprisingly, some children with the condition are healthy and develop normally.3 1... 

He was provided with a diet restricted in protein but was found to limit his own protein intake to 1.0 to 1.2 g/kg. Treatment with large doses of vitamin B12 for a period of 4 months made no difference to the concentration of methylmalonic acid excreted in his urine. He required frequent hospitalization for anorexia, vomiting, and dehydration. At 18 months of age he developed renal failure, and at 3 years he became oliguric. His physical development appeared normal for the first 12 months and then it deteriorated. He started to walk at 31 years, but at this time he also had hepatomegaly and persistent vomiting. On the basis of a developmental screening test he was found to be 12 to 18 months behind in all areas tested. 

A 6-month-old child was breast-fed exclusively by a mother who had been a strict vegetarian for at least 7 years. He was totally unresponsive to stimuli. His hemoglobin was 5.7 g/dL, and his bone marrow aspirates showed megaloblastic changes in blood cells. His serum folate and iron were normal. His urine contained increased amounts of homocystine, methylmalonic acid, and glycine. Propose a reason for this infant s illness, and discuss its biochemical etiology. Discuss other possible reasons for the same or similar symptoms in a patient. Explain the abnormal serum and urine chemistries. 

Indirect indicators of vitamin B12 deficiency include measurements of the metabolites homocysteine and methylmalonic acid (MMA) in serum and MMA in urine (see the Biochemical Perspectives section). Whereas the serum homocysteine concentration increases during folate or vitamin B12 deficiencies, the serum and urine MMA concentrations increase only in vitamin B12 deficiency. Therefore, MMA determinations can be used to differentiate vitamin B12 deficiency from folate deficiency. The normal concentration of MMA in serum ranges from 0.08 to 0.28 pmol/L. MMA is quantified using gas-liquid chromatography and mass spectrometry. Elevated concentrations of MMA and homocysteine in serum may precede the development of hematological abnormalities and reductions in serum vitamin B12 concentrations. One should be aware that other conditions, including renal in sufficiency and inborn errors of metabolism, can also result in elevated serum levels of MMA. 

As a result of the reduced activity of the mutase in vitamin B12 deficiency, there is an accumulation of methyhnalonyl CoA, some of which is hydrolyzed to yield methylmalonic acid, which is excreted in the urine. As discussed in Section 10.10.3, this can be exploited as a means of assessing vitamin B12 nutritional status. There may also be some general metabolic acidosis, which has been attributed to depletion of CoA because of the accumulation of methyl-malonyl CoA. However, vitamin B12 deficiency seems to result in increased synthesis of CoA to maintain normal pools of metabolically useable coenzyme. Unlike coenzyme A and acetyl CoA, neither methylmalonyl CoA nor propionyl CoA (which also accumulates in vitamin B12 deficiency) inhibits pantothenate kinase (Section 12.2.1). Thus, as CoA is sequestered in these metabolic intermediates, there is relief of feedback inhibition of its de novo synthesis. At the same time, CoA may be spared by the formation of short-chain fatty acyl carnitine derivatives (Section 14.1.1), which are excreted in increased amounts in vitamin B12 deficiency. In vitamin Bi2-deficient rats, the urinary excretion of acyl carnitine increases from 10 to 11 nmol per day to 120nmolper day (Brass etal., 1990). 

The initial dose in cobalamin deficiency anaemias, including uncomplicated pernicious anaemia, is hydroxocobalamin 1 mg i.m. every 2-3 days for 5 doses to induce remission and to replenish stores. Maintenance may be 1 mg every 3 months higher doses will not find binding sites and will be eliminated in the urine. Higher doses are justified during renal or peritoneal dialysis where hydroxy-cobalamin clearance is increased, and resultant raised plasma methylmalonic acid and homocysteine represent an independent risk factor for vascular events in these patients (see later). 

The data in Table 9.2 concern a study in rats. l he animals w cre raised on nutritionally complete diets or vitamin Bn-deficienl diets for 3 months. Then urine was collect over the course of a day and used for analysis of metabolites related to folate and vitamin Bx2 (FIGLU and methylmalonic acid). The data show that Bu deficiency induces a dramatic increase in urinary FIGLU. Rats do not requite a... 

Breakdown of isoleucine, valine, threonine, and methionine results in the production of propionyl-CoA. Propionyl-CoA, in turn, is catabolized to succinyl-CoA via the intermediate methylmalonyl-CoA. Methylmalonyl-CoA is a compoimd of imusual interest to nutritional scientists. This compound accumulates in the cell during a vitamin B12 deficiency. Vitamin B12 deficiency is not a rare disease, as it appears in a common autoimmune disease called pernicious anemia. Vitamin B12 deficiency also occurs in strict vegetarians who avoid meat, fish, poultry, and dairy products. Methylmalonyl-CoA can also build up with rare genetic diseases that involve the production of defective, mutant forms of methylmalonyl-CoAmutase. Most of the methylmalonyl-CoAthat accumulates to abnormally high levels in the cell is hydrolyzed to methylmalonic acid (MMA), which leaves the cell for the bloodstream and eventual excretion in the urine. Some of the MMA is converted back to propionyl-CoA, resulting in the production and accumulation of propionic acid in the cell. The measurement of plasma and urinary MMA has proven to be a method of choice for the diagnosis of vitamin B12 deficiency, whether induced by pernicious anemia or by dietary deficiency. 

Ethylmalonic Acid. Stalder (S35) found ethylmalonic acid in normal urine of rat and man and showed that its excretion by the rat increases when isoleucine is fed. The increase is analogous to that of methylmalonic acid when valine is fed. 

In dietary liver necrosis in rats, methylmalonic acid is greatly increased in the urine, representing 70 % of total ether-soluble adds instead of 10 % as in normal animals a,a-dimethylsuccinie add is also increased, but to a smaller extent (B3, F6). 

---